tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page a
Technical Manual
PolyATtract® mRNA
Isolation Systems
INSTRUCTIONS FOR USE OF PRODUCTS Z5200, Z5210, Z5300 AND
Z5310.
IMPORTANT NOTICE
The magnet used with this system generates a very strong
magnetic field. Do not place the magnet near computer screens,
diskettes, pacemakers or other electronic equipment.
Note: Due to its strong attraction to metal objects, the magnet may
chip upon sudden hard impact with such objects. The chips could
potentially cause eye injury. Wear protective eyewear when
handling this unit.
If you have additional questions, please contact Promega
Technical Services by phone at 800-356-9526 or by e-mail:
[email protected].
PRINTED IN USA.
Revised 12/12
Part# TM021
tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page 1
PolyATtract® mRNA
Isolation Systems
All technical literature is available on the Internet at: www.promega.com/protocols/
Please visit the web site to verify that you are using the most current version of this
Technical Manual. Please contact Promega Technical Services if you have questions on use
of this system. E-mail: [email protected]
1. Description..........................................................................................................2
2. Product Components and Storage Conditions ............................................4
3. Creating a Ribonuclease-Free Environment.................................................5
4. Protocol for Large-Scale mRNA Isolation:
PolyATtract® Systems I and II ........................................................................6
A.
B.
C.
D.
E.
Annealing of Probe ..............................................................................................6
Stock Solution Preparation..................................................................................6
Washing of Streptavidin-Paramagnetic Particles............................................6
Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids...................7
Elution of mRNA..................................................................................................7
5. Protocol for Small-Scale mRNA Isolation:
PolyATtract® Systems III and IV....................................................................8
A.
B.
C.
D.
E.
Annealing of Probe ..............................................................................................8
Stock Solution Preparation..................................................................................8
Washing of Streptavidin-Paramagnetic Particles............................................8
Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids...................9
Elution of mRNA..................................................................................................9
6. Analysis and Handling of Purified mRNA................................................10
A. Determining mRNA Concentration and Purity ............................................10
B. Precipiting and Concentrating mRNA for Secondary Applications ..........12
7. Troubleshooting...............................................................................................13
8. References .........................................................................................................13
9. Appendix ...........................................................................................................14
A. Composition of Buffers and Solutions ............................................................14
B. Related Products.................................................................................................14
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 1
tm021.1212:EIVD_TM.qxd
1.
12/18/2012
8:38 AM
Page 2
Description
The PolyATtract® mRNA Isolation Systems use the MagneSphere® technology
to eliminate the need for oligo(dT) cellulose and its associated problems. With
total RNA as the starting material, the poly(A) mRNA fraction can be isolated
free of other nucleic acid contamination in approximately 45 minutes. The
isolated mRNA is suitable for all molecular biology applications, including
in vitro translation and cDNA synthesis.
The systems use a biotinylated oligo(dT) primer to hybridize at high efficiency
in solution to the 3´ poly(A) region present in most mature eukaryotic mRNA
species. The hybrids are captured and washed at high stringency using
streptavidin coupled to paramagnetic particles and a magnetic separation stand
(Figure 1). The mRNA is eluted from the solid phase by the simple addition of
ribonuclease-free deionized water. This procedure yields an essentially pure
fraction of mature mRNA after only a single round of magnetic separation.
total RNA containing
mRNA fraction
AAAAAAA3 ′
5′
m7G
Hybridize with
biotin-oligo(dT).
5′
B-T T T T T T T 3′
AAAAAAA3 ′
3′
T T T T T T T-B5′
5′
m7G
Add streptavidin PMPs.
PMP
AAAAAAA3 ′
T T T T T T T-B
5′
m7G
PMP
PMP
Wash and elute.
AAAAAAA3 ′
5′
m7G
T T T T T T T-B
(aqueous)
(solid)
PMP
0369MA03_1A
m7G
MAGNET
AAAAAAA3 ′
T T T T T T T-B
5′
MAGNET
Magnetize.
Figure 1. Schematic diagram of the PolyATtract® mRNA isolation procedure.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 2
Printed in USA.
Revised 12/12
tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page 3
All components in the system are guaranteed to be free of contaminating
ribonucleases when used as directed and are thoroughly tested to ensure
optimal performance. When used in combination with the SV Total RNA
Isolation System, a pure fraction of intact, full-length mRNA can be isolated
from a tissue or cell source in as little as 2 hours.
The PolyATtract® mRNA Isolation Systems are available in two basic
configurations, which depend on the amount of input RNA. PolyATtract®
Systems I and II are designed for larger amounts of input RNA; either system
provides reagents sufficient for three isolations, each from 1–5mg of total RNA.
PolyATtract® System I does not include a magnetic separation stand;
PolyATtract® System II includes the stand.
PolyATtract® Systems III and IV provide the MagneSphere® Streptavidin
Paramagnetic Particles in smaller aliquots, suitable for use with smaller
amounts of input RNA. Each of these systems contains reagents sufficient to
perform fifteen mRNA isolations, starting with 1mg or less total RNA per
isolation.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 3
tm021.1212:EIVD_TM.qxd
2.
12/18/2012
8:38 AM
Page 4
Product Components and Storage Conditions
Product
Cat.#
PolyATtract® mRNA Isolation System I (Refill for System II)
Z5210
Each system contains all the reagents and RNase-free tubes required (excluding the
Magnetic Separation Stand) to perform three separate mRNA isolations, each from
1–5mg of total RNA. See Cat.# Z5200.
Product
Cat.#
PolyATtract® mRNA Isolation System II
Z5200
Each system contains all the reagents and RNase-free tubes required to perform three
separate mRNA isolations, each from 1–5mg of total RNA. Includes:
•
•
•
•
•
•
35µl
4.2ml
9ml
75ml
3 each
1 each
Biotinylated Oligo(dT) Probe (50pmol/µl)
20X SSC Solution (3 × 1.4ml)
Streptavidin MagneSphere® Paramagnetic Particles (3 × 3ml)
Nuclease-Free Water (3 × 25ml)
mRNA User Tubes
MagneSphere® Magnetic Separation Stand for 12 × 75mm Tubes
Product
Cat.#
PolyATtract® mRNA Isolation System III
Z5300
Each system contains all the reagents required to perform fifteen separate mRNA
isolations, each from up to 1mg of total RNA. Includes:
•
•
•
•
•
50µl
2.8ml
9ml
50ml
1 each
Biotinylated Oligo(dT) Probe (50pmol/µl)
20X SSC Solution (2 × 1.4ml)
Streptavidin MagneSphere® Paramagnetic Particles (15 × 0.6ml)
Nuclease-Free Water (2 × 25ml)
MagneSphere® Magnetic Separation Stand for 1.5ml
Microcentrifuge Tubes
Product
Cat.#
PolyATtract® mRNA Isolation System IV (Refill for System III)
Z5310
Each system contains all the reagents required (excluding the Magnetic Separation
Stand) to perform fifteen separate mRNA isolations, each from up to 1mg of total RNA.
See Cat.# Z5300.
Storage and Stability: If handled and stored properly, the performance of this
product is guaranteed for at least 6 months from the date of purchase unless
otherwise stated on the label. Store at 4°C.
!
Do not freeze the Streptavidin MagneSphere® Particles, as this will reduce
their performance.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 4
Printed in USA.
Revised 12/12
tm021.1212:EIVD_TM.qxd
3.
12/18/2012
8:38 AM
Page 5
Creating a Ribonuclease-Free Environment
The following notes will help you prevent the accidental contamination of
samples with ribonuclease (RNase), allowing the isolation of full-length mRNA.
1.
Two of the most common sources of RNase contamination are the user’s
hands and bacteria and molds that may be present on airborne dust
particles. To prevent this type of contamination, proper microbiological
sterile technique should be observed when handling the reagents supplied
with the system. The reagents provided will be used repeatedly, so
particular care must be taken to prevent contamination when opening
and closing reagent tubes. Gloves should be worn at all times.
2.
Whenever possible, sterile disposable plasticware should be used to
handle RNA. These materials are generally RNase-free and thus do not
require pretreatment to inactivate RNase.
3.
Nondisposable glass and plasticware should be treated before use to
ensure that it is RNase-free. Glassware should be baked at 200°C
overnight, and plasticware should be thoroughly rinsed before use with
0.1N NaOH, 1mM EDTA, followed by RNase-free water. COREX® tubes
should be rendered RNase-free by treatment with diethyl pyrocarbonate
(see below) and not by baking. This will reduce the chance of tube failure
during centrifugation.
4.
Solutions supplied by the user should be treated with 0.05% diethyl
pyrocarbonate (DEPC) overnight at room temperature, then autoclaved
for 30 minutes to remove any trace of DEPC. Likewise, glassware can be
rinsed in 0.05% DEPC overnight, then autoclaved to remove residual
DEPC.
!
Tris solutions cannot be treated with DEPC.
Note: Many sources of distilled water are free of contaminating RNase
activity. Test your water source for the presence of contaminating RNase.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 5
tm021.1212:EIVD_TM.qxd
4.
12/18/2012
8:38 AM
Page 6
Protocol for Large-Scale mRNA Isolation: PolyATtract® Systems I and II
This procedure is designed for use with 1–5mg of total RNA.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 9.A.)
• 65°C water bath or heating block
• sterile, RNase-free plastic tubes
• sterile, RNase-free pipets and pipette tips
4.A. Annealing of Probe
1. In a sterile, RNase-free 3ml tube, combine 1–5mg of total RNA and RNaseFree Water to a final volume of 2.43ml.
2. Place the tube in a 65°C heating block for 10 minutes.
3. Add 10µl of the Biotinylated-Oligo(dT) Probe and 60µl of 20X SSC to the
RNA. Mix gently, and incubate at room temperature until completely
cooled. This may require up to 30 minutes, depending on the size of the
tube. While this solution is cooling, prepare stock solutions of 0.5X and
0.1X SSC.
4.B. Stock Solution Preparation
1. Prepare 5ml of sterile 0.5X SSC by combining 0.125ml of 20X SSC with
4.875ml of RNase-Free Water in a sterile, RNase-free tube.
2. Prepare 10ml of sterile 0.1X SSC by combining 50µl of 20X SSC with 9.95ml
of RNase-Free Water in a sterile, RNase-free tube.
4.C. Washing of Streptavidin-Paramagnetic Particles
The Streptavidin MagneSphere® Paramagnetic Particles require bovine serum
albumin (BSA) for stabilization, which is present in the storage buffer and is
removed once the particles are washed. The SA-PMPs are provided at a
concentration of 1mg/ml in a solution of PBS, 1mg/ml BSA and 0.02% sodium
azide. The particles should be rinsed three times each with an equal volume of
0.5X SSC and used within 30 minutes after washing to maintain optimal
performance. The particles cannot be washed and reused after the initial use.
The particless must be completely resuspended to ensure adequate
performance. Discard particles that appear to have “clumped” and cannot be
dispersed. To determine if the particles are in good condition, mix by inverting
the tube several times and verify that the particles remain in suspension for at
least 3 minutes in a 0.5–1ml volume. If some of the particles settle out of
suspension within 3 minutes, forming an easily visible pellet, they should not
be used. To prevent clumping of the particles, do not freeze them or allow
them to dry out.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 6
Printed in USA.
Revised 12/12
tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page 7
1. Resuspend one tube (3ml volume) of the Streptavidin MagneSphere®
Paramagnetic Particles (SA-PMPs) per isolation by gently flicking the
bottom of the tube until they are completely dispersed, then capture them
by placing the tube in the magnetic stand until the SA-PMPs have collected
at the side of the tube (approximately 30 seconds).
2. Carefully remove the supernatant. Do not centrifuge the particles.
3. Wash the SA-PMPs three times with 0.5X SSC (1.5ml per wash), each time
capturing them using the magnetic stand and carefully removing the
supernatant.
4. Resuspend the washed SA-PMPs in 0.5ml of 0.5X SSC.
4.D. Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids
1. Add the entire contents of the annealing reaction (Section 4.A, Step 3) to
the tube containing the washed SA-PMPs.
2. Incubate at room temperature for 10 minutes. Gently mix by inverting
every 1–2 minutes.
3. Capture the SA-PMPs using the magnetic stand, and carefully remove the
supernatant without disturbing the SA-PMP pellet.
Note: Save the supernatant from Step 3 until you are certain that
satisfactory binding and elution of mRNA has occurred.
4. Wash the particles four times with 0.1X SSC (1.5ml per wash) by gently
flicking the bottom of the tube until all particles are resuspended. After the
final wash, remove as much of the supernatant as possible without
disturbing the SA-PMPs.
4.E. Elution of mRNA
1. Resuspend the final SA-PMP pellet (Section 4.D, Step 4) in 1.0ml of the
RNase-Free Water, and gently resuspend the particles by flicking the tube.
2. Magnetically capture the SA-PMPs, and transfer the eluted mRNA to one
of the 2ml User Tubes provided. Do not discard the particles.
Note: If particles have been transferred with the eluted mRNA, remove by
centrifuging at 12,000 × g for 1 minute. Carefully transfer the RNA to a new
RNase-free tube.
Refer to Section 6 for recommended procedures to analyze and handle the
purified mRNA.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 7
tm021.1212:EIVD_TM.qxd
5.
12/18/2012
8:38 AM
Page 8
Protocol for Small-Scale mRNA Isolation: PolyATtract® Systems III and IV
This procedure is designed for use with up to 1mg of total RNA.
Materials to Be Supplied by the User
(Solution compositions are provided in Section 9.A.)
• 65°C water bath or heating block
• sterile, RNase-free plastic tubes, 1.5ml
• sterile, RNase-free pipets and pipette tips
5.A. Annealing of Probe
1. In a sterile, RNase-free 1.5ml tube, bring 0.1–1.0mg of total RNA to a final
volume of 500µl in RNase-Free Water.
Note: Less total RNA (50µg) may be used, but the mRNA obtained may not
be detectable by spectrophotometry (Section 6).
2. Place the tube in a 65°C heating block for 10 minutes.
3. Add 3µl of the Biotinylated-Oligo(dT) Probe and 13µl of 20X SSC to the
RNA. Mix gently, and incubate at room temperature until completely
cooled. This should require 10 minutes or less. While this solution is
cooling, prepare stock solutions of 0.5X and 0.1X SSC.
5.B. Stock Solution Preparation
1. Prepare 1.2ml of sterile 0.5X SSC by combining 30µl of 20X SSC with
1.170ml of RNase-Free Water in a sterile, RNase-free tube.
2. Prepare 1.4ml of sterile 0.1X SSC by combining 7µl of 20X SSC with 1.393ml
of RNase-Free Water in a sterile, RNase-free tube.
5.C. Washing of Streptavidin-Paramagnetic Particles
The Streptavidin MagneSphere® Paramagnetic Particles require BSA for
stabilization, which is present in the storage buffer and is removed once the
particles are washed. The SA-PMPs are provided at a concentration of 1mg/ml
in a solution of PBS, 1mg/ml BSA and 0.02% sodium azide. The particles
should be rinsed three times each with an equal volume of 0.5X SSC and used
within 30 minutes after washing to maintain optimal performance. The
particles cannot be washed and reused after the initial use.
The particles must be completely resuspended to ensure adequate
performance. Discard particles that appear to have “clumped” and cannot be
dispersed. To determine if the particles are in good condition, mix by inverting
the tube several times and verify that the particles remain in suspension for at
least 3 minutes in a 0.5–1ml volume. If some of the particles settle out of
suspension within 3 minutes, forming an easily visible pellet, they should not
be used. To prevent clumping of the particles, do not freeze them or allow
them to dry out.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 8
Printed in USA.
Revised 12/12
tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page 9
1. Resuspend one tube (0.6ml volume) of the Streptavidin MagneSphere®
Paramagnetic Particles (SA-PMPs) per isolation by gently flicking the
bottom of the tube until they are completely dispersed, then capture them
by placing the tube in the magnetic stand until the SA-PMPs have collected
at the side of the tube (approximately 30 seconds).
2. Carefully remove the supernatant. Do not centrifuge the particles.
3. Wash the SA-PMPs three times with 0.5X SSC (300µl per wash), each time
capturing them using the magnetic stand and carefully removing the
supernatant.
4. Resuspend the washed SA-PMPs in 100µl of 0.5X SSC.
5.D. Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids
1. Add the entire contents of the annealing reaction (Section 5.A., Step 3) to
the tube containing the washed SA-PMPs.
2. Incubate at room temperature for 10 minutes. Gently mix by inverting
every 1–2 minutes.
3. Capture the SA-PMPs using the magnetic stand, and carefully remove the
supernatant without disturbing the SA-PMP pellet.
Note: Save the supernatant from Step 3 until you are certain that
satisfactory binding and elution of mRNA has occurred.
4. Wash the particles four times with 0.1X SSC (300µl per wash) by gently
flicking the bottom of the tube until all of the particles are resuspended.
After the final wash, remove as much of the supernatant as possible
without disturbing the SA-PMPs.
5.E. Elution of mRNA
1. Resuspend the final SA-PMP pellet (Section 5.D, Step 4) in 100µl of the
RNase-Free Water, and gently resuspend the particles by flicking the tube.
2. Magnetically capture the SA-PMPs, and transfer the eluted mRNA to a
sterile, RNase-free tube. Do not discard the particles.
3. Repeat the elution step by resuspending the SA-PMP pellet in 150µl of
RNase-Free Water. Repeat the capture step, pooling the eluate with the
RNA eluted in Section 5.E., Step 2 (250µl total volume).
Note: If particles have been transferred with the mRNA, remove by
centrifuging at 12,000 × g for 1 minute. Carefully transfer the RNA to a
new RNase-free tube.
Refer to Section 6 for recommended procedures to analyze and handle the
purified mRNA.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 9
tm021.1212:EIVD_TM.qxd
6.
12/18/2012
8:38 AM
Page 10
Analysis and Handling of Purified mRNA
6.A. Determining mRNA Concentration and Purity
The concentration and purity of eluted mRNA can be determined by
spectrophotometry. Pure mRNA has an A260/A280 absorbance ratio of ≥2.0. To
estimate the mRNA concentration, assume that a 40µg/ml mRNA solution has
an absorbance of 1 at 260nm. Typically, 1–5% of total RNA is mRNA (1).
Semi-micro or micro cell cuvettes should be used for sample volumes of 1ml
or less. Micro Short Cells (25mm tall) can be used to measure the absorbance
of 300–400µl samples, and 50–300µl sample absorbances can be measured in
96-well UV spectrophotometers using UV translucent plates and pathlength
correction values. The minimum volume that can be measured in the cuvette
depends upon the position of the light beam in the instrument. Refer to the
owner’s manual, or contact the manufacturer. Shorter pathlength cuvettes
can be used to measure smaller volumes, but according to Beer’s Law
(ε × pathlength × concentration), a shorter pathlength requires a higher
concentration of mRNA to give meaningful absorbance values. The standard
pathlength of a cuvette is 1cm. Sub-micro cells are available to measure
sample volumes as small as 10µl. The compatibility of these cells with your
spectrophotometer needs to be determined. Black masked cuvettes are
preferable to clear-wall microcuvettes for a lower signal:noise ratio. The
window material of the cuvette should be polished quartz with a usable
wavelength of <200nm (e.g., Starna® Spectrosil Far UV Quartz). Polystyrene
and acrylic cuvettes are not suitable for measurement in the 230–280nm
wavelength range unless specified.
Note: Be certain that cuvettes are RNase-free so that samples can be recovered
after spectrophotometry. This can be done by washing the cuvettes briefly in
50mM NaOH followed by rinsing with sterile water.
The quality of isolated mRNA also may be checked by denaturing agarose gel
electrophoresis (1), but this may require the entire sample to be loaded when
<1mg of total RNA is used for mRNA isolation. These gels can be saved and
used for Northern blots. Figure 2, Panel A, illustrates the appearance of the
mRNA fraction isolated from total mouse liver RNA using the PolyATtract®
System. The mRNA should appear as a smear extending from approximately
8.0kb to approximately 0.5kb (depending on the tissue). The bulk of the
mRNAs should be clustered around 2.0kb.
We normally see very little ribosomal contamination after the first round of
selection. However, the appearance of some ribosomal bands does not indicate
poor performance of the system. Figure 2, Panel B, shows a Northern blot of
mRNA, which contained visible amounts of both 28S and 18S ribosomal
RNAs. The blot was probed with 32P-labeled biotinylated oligo(dT). Little or
no hybridization is seen in the total RNA lane, whereas the mRNA-selected
material shows a tremendous enrichment despite the presence of some
ribosomal RNAs. Therefore, a small amount of ribosomal contamination
should not affect the functionality of the mRNA in most applications.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 10
Printed in USA.
Revised 12/12
– Markers
– Total RNA
Page 11
– PolyATtract® mRNA
8:38 AM
– Markers
– Total RNA
12/18/2012
– PolyATtract® mRNA
tm021.1212:EIVD_TM.qxd
– 7.5kb
– 7.5kb
– 4.4kb
– 4.4kb
– 2.4kb
– 2.4kb
– 1.4kb
– 1.4kb
0431TA12_2A
– 0.2kb
– 0.2kb
Panel A
Panel B
Figure 2. Northern blot of purified mRNA using the PolyATtract® mRNA
Isolation System. Panel A. Ethidium bromide-stained RNA samples (5µg per lane)
on a 1% denaturing agarose gel. Lane 1, mRNA fraction; lane 2, total mouse liver
RNA; lane 3, 0.24–7.5kb RNA molecular weight standards. Panel B. RNAs in
Panel A were blotted to nitrocellulose and probed with 32P-labeled biotinylated
oligo(dT) at 50°C in 6X SSC containing 1X Denhardt's solution and 0.1% SDS.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 11
tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page 12
6.B. Precipitating and Concentrating mRNA for Secondary Applications
While the mRNA fraction isolated with the PolyATtract® System may be
sufficiently concentrated for spectrophotometric analysis, it may be too dilute
for applications such as cDNA cloning and translation in vitro. If greater than
0.5mg of total RNA is used for mRNA isolation, the RNA may be concentrated
by alcohol precipitation (2):
1. For cDNA cloning: Add 0.1 volume of 3M sodium acetate (pH 5.2) and
1.0 volume of isopropanol to the eluate, then incubate at –20°C overnight.
For translation in vitro: Add 0.1 volume of 3M potassium or ammonium
acetate and 1.0 volume of isopropanol to the eluate, then incubate at –20°C
overnight.
2. Centrifuge at >12,000 × g for 10 minutes. Resuspend the RNA pellet in 1ml
of 75% ethanol, and centrifuge again.
3. For short-term storage: Dry the pellet in a vacuum desiccator for about
15 minutes, resuspend in RNase-free, deionized water at 0.5–1.0µg/µl and
store at –70°C.
For long-term storage: Store the RNA in sodium acetate/isopropanol
solution at –70°C, and centrifuge just prior to use.
When less than 0.5mg of total RNA is used as starting material, the theoretical
yields of mRNA generally are expected to be low, and precipitation becomes
an inefficient means of recovery. For low-yield samples, freeze the mRNA
eluate at –20°C for 10 minutes, then dry down in a Speed Vac®. This will take
approximately 2.5 hours. Samples may be rehydrated in a nominal volume for
cDNA cloning or in vitro translation. In our hands, a cDNA library can be
made with mRNA isolated from as little as 100µg of total RNA.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 12
Printed in USA.
Revised 12/12
tm021.1212:EIVD_TM.qxd
7.
12/18/2012
8:38 AM
Page 13
Troubleshooting
For questions not addressed here, please contact your local Promega Branch Office or Distributor.
Contact information available at: www.promega.com. E-mail: [email protected]
Symptoms
Causes and Comments
No mRNA eluted
No mRNA was bound because salt was omitted
from annealing step. Repeat annealing step,
adding 20X SSC (to 0.5X final).
Insufficient cooling of annealing reaction before
probe capture and wash. Add the saved
supernatant back to the particles as in
Sections 4.D and 5.D, Step 1, and continue
the procedure.
Salt was not eliminated before elution. Wash
final SA-PMP pellet again with deionized water,
and check the A260 of this eluate.
RNase contamination in total RNA. Evaluate
quality of total RNA by gel electrophoresis, and
repeat total RNA isolation as necessary.
RNA appears degraded on gel
RNase contamination during mRNA isolation.
Repeat entire procedure. Reread Section 3.
Low yield
High-stringency wash. Repeat annealing step,
adding 20X SSC (to 1.0X final), and perform the
final wash step with 0.2X SSC.
8.
References
1. Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY.
2. Wallace, D.M. (1987) Precipitation of nucleic acids. Meth. Enzymol. 152, 41–8.
Additional References
1. Barron, D. (1998) Technically Speaking: Streptavidin MagneSphere® Paramagnetic
Particles. Promega Notes 66, 17–8.
2. Marcus, L. et al. (1996) PolyATtract® Systems for mRNA purification. Promega Notes
60, 14–8.
3. Burke, P. (1996) Technically Speaking: PolyATtract® mRNA Isolation Systems.
Promega Notes 56, 27–9.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 13
tm021.1212:EIVD_TM.qxd
9.
12/18/2012
8:38 AM
Page 14
Appendix
9.A. Composition of Buffers and Solutions
The performance of this system is guaranteed when used with the buffers provided
with the system. For users who wish to make their own buffers, it is important
that all reagents and equipment used are RNase-free (see Section 3).
20X SSC
3M sodium acetate (pH 5.2)
87.7g NaCl
44.1g sodium citrate1
Dissolve in 400ml of Nuclease-Free
Water. Adjust pH to 7.2 with HCl, and
bring the volume to 500ml. Dispense
into aliquots. Sterilize by autoclaving.
1Trisodium
408.1g sodium acetate • 3H2O
Dissolve sodium acetate in 800ml of
water. Adjust pH to 5.2 with glacial
acetic acid. Adjust the volume to 1 liter
with water. Sterilize by autoclaving.
salt dihydrate
9.B. Related Products
Systems for Total RNA Isolation
Product
SV 96 Total RNA Isolation System
Size
1× 96 each
5× 96 each
SV Total RNA Isolation System
10 preps
50 preps
250 preps
Vacuum Adapters
20 each
®
Vac-Man Laboratory Vacuum Manifold, 20-sample capacity
1 each
Red Blood Cell Lysis Solution (CLB)
200ml
RNA Lysis Buffer (RLA)
50ml
Cat.#
Z3500
Z3505
Z3101
Z3100
Z3105
A1331
A7231
Z3141
Z3051
Systems for mRNA Isolation Directly from Biological Samples
Product
PolyATtract® System 1000
with Magnetic Separation Stand
without Magnetic Separation Stand
PolyATtract® System 1000 Magnetic Separation Stand
Cat.#
Scalable
Scalable
1 each
Z5420
Z5400
Z5410
Each system contains sufficient reagents to isolate RNA from up to 2g of tissue or 4 × 108
cultured cells.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Part# TM021
Page 14
Printed in USA.
Revised 12/12
tm021.1212:EIVD_TM.qxd
12/18/2012
8:38 AM
Page 15
Amplification-Related Products
Product
Access RT-PCR System
Size
500 reactions
100 reactions
Access RT-PCR Introductory System
20 reactions
PCR Master Mix
100 reactions
1,000 reactions
GoTaq® DNA Polymerase†
100u
500u
2,500u
M-MLV Reverse Transcriptase
10,000u
M-MLV Reverse Transcriptase, RNase H Minus**
10,000u
M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant** 10,000u
ImProm-II™ Reverse Transcriptase
100 reactions
500 reactions
AMV Reverse Transcriptase
300u
®
Recombinant RNasin Ribonuclease Inhibitor
2,500u
®
RNasin Plus RNase Inhibitor
2,500u
Cat.#
A1280
A1250
A1260
M7502
M7505
M3001
M3005
M3008
M1701
M5301
M3682
A3802
A3803
M5101
N2511
N2611
†Catalog
numbers may be different in Europe.
**This product is not available for purchase in the United States.
Other Related Products
Product
Biotinylated Oligo(dT) Probe (50pmol/µl)
Streptavidin MagneSphere® Paramagnetic Particles
RNA Markers
Size
35µl
9ml (15 × 0.6ml)
25ml
50µl
Cat.#
Z5261
Z5481
Z5482
G3191
© 1991–2012 Promega Corporation. All Rights Reserved.
GoTaq, MagneSphere, PolyATtract, RNasin and Vac-Man are registered trademarks of Promega Corporation. ImProm-II is a
trademark of Promega Corporation.
COREX is a registered trademark of Corning, Inc. Speed Vac is a registered trademark of Savant Instruments, Inc. Starna is a
registered trademark of Starna Cells, Inc.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more
information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the
most up-to-date information on Promega products.
Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA
Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com
Printed in USA.
Revised 12/12
Part# TM021
Page 15